Fluorescent dyes are widely used as tracers for localization of biological structures by fluorescence microscopy, for quantitation of analytes by fluorescence immunoassay, for flow cytometric analysis of cells, for measurement of physiological state of cells and other applications [Kanaoka (1977); Hemmila (1985)]. Their primary advantages over other types of absorption dyes include the visibility of emission at a wavelength distinct from the excitation, the orders of magnitude greater detectability of fluorescence emission over light absorption, the generally low level of fluorescence background in most biological samples and the measurable intrinsic spectral properties of fluorescence polarization [Jolley, et al. (1981)], lifetime and excited state energy transfer (U.S. Pat. No. 3,996,345).
For many applications that utilize fluorescent dyes as tracers, it is necessary to chemically react the dye with a biologically active ligand such as a cell, tissue, protein, antibody, enzyme, drug, hormone, nucleotide, nucleic acid, polysaccharide, lipid or other biomolecule to make a fluorescent ligand analog or with natural or synthetic polymers. With these synthetic probes the biomolecule frequently confers a specificity to a biochemical interaction that is to be observed and the fluorescent dye provides the method for detection and/or quantitation of the interaction. Chemically reactive synthetic fluorescent dyes have long been recognized as essential for following these interactions [Soini and Hemmila, 1979]. The dyes in common use are limited to a relatively small number of aromatic structures. It is the object of this invention to provide improved fluorescent dyes. It is further an object of this invention to provide dyes with the chemical reactivity necessary for conjugation to the functional groups commonly found in biomolecules, drugs, and natural and synthetic polymers.
Coons and Kaplan in 1950 first prepared a chemically reactive isocyanate of fluorescein and later Riggs, et al. (1958) introduced the more stable isothiocyanate analog of fluorescein. Fluorescein isothiocyanate (FITC) remains one of the most widely used tracers for fluorescent staining and immunoassay. Other reactive fluoresceins were prepared by Haugland (U.S. Pat. No. 4,213,904). Virtually all fluorescence microscopes are equipped with excitation sources and filters optimized to excite and detect fluorescein emission. Due to the intense but discrete excitation of the argon laser at 488 nm which is strongly absorbed by the primary fluorescein absorption band (maximum at 492 nm), fluorescein has also become the primary dye for use in the technique of flow cytometry [Lanier & Loken (1984)].
The primary advantages that have permitted fluorescein isothiocyanate and its conjugates to remain the standard for microscopy and fluorescence immunoassay are a high absorbance, a high quantum yield and general ease of conjugation to biomolecules. The only fluorescent tracers in common use that exceed the overall fluorescence yield on a molar basis are the phycobiliproteins [U.S. Pat. No. 4,520,110; Oi, et al (1983), Kronick (1983)]. These require special methods for conjugate to biomolecules and in some cases such as fluorescence polarization immunoassays [Jolley, et al. (1981)] have too high a molecular weight to be useful. They also have high susceptibility to photodegradation. The only chemically reactive fluorophores with similar spectra to fluorescein that have been described are derived from the nitrofurazan structure [Soini and Hemilla (1979)]. These fluorophores have much weaker absorptivity (less than 25,000 cm.sup.-1 M.sup.-1 at its peak at 468 NM versus 75,000 cm.sup.-1 M.sup.-1 for fluorescein at its peak near 490 NM) and virtually no fluorescence in aqueous solutions where fluorescein is usually used and where most applications in immunofluorescence exist.
Despite their widespread acceptance as fluorescent tracers, fluorescein tracers have some deficiencies that preclude or make more difficult some useful applications. Primary is the strong tendency of the fluorophore to photobleach when illuminated by a strong excitation source such as the lamp used in fluorescence microscopes. The photobleaching can result in a significant percentage of the fluorescence being lost within seconds of the onset of illumination. In fluorescence microscopy this results in loss of the image. In fluorescence assays, the loss of fluorescence with time makes quantitation of results more difficult and ultimately decreases the sensitivity of detection of the analyte. To a variable degree, extrinsic reagents including propyl gallate and p-phenylenediamine retard but do not eliminate the photobleaching. Additionally, fluorescein shows a pH dependent absorption spectrum which decreases the fluorescence yield in solutions at a physiological pH or below. Furthermore, the emission spectrum of fluorescein has a very broad long wavelength component which greatly increases its background at wavelengths used for detection of other dyes such as rhodamine B in applications such as DNA sequencing [L. M. Smith, et al. (1986)] and flow cytometry that require detection of multiple dyes. Due to the chemical nature of fluorescein, all of its fluorescent derivatives have an ionic charge. The ionic charge and general lack of solubility in non-polar solvents preclude its use as a fluorescent tracer for lipophilic structures and decrease its suitability or such applications as a chromatographic derivatization reagent.